The subject of the invention is also physiologically acceptable compositions comprising the said compounds.
The subject of the invention is also the use of the said compounds in the preparation of drugs intended for man or animals.
The important physiological role of endogenous neuropeptides, and in particular that of cholecystokinin (CCK), is known. This neuropeptide is both a digestive hormone and a neurotransmitter in the central and peripheral nervous systems. In the latter case, it is mainly in the form of an octapeptide sulphate (CCK-8-S) corresponding to the formula:
Asp-Tyr (SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2, (SEQ ID NO: 7)
CCK exerts various actions on motricity and digestive secretions (contraction of the bile vesicle, inhibition of gastric secretion, etc.) as well as on the central nervous system (analgesia, action on moods and cognition) and on the endocrine system (hypophyseal secretions). In addition, CCK and certain derivatives have a powerful anorexigenic activity by facilitating satiation by means of stimulating peripheral, and possibly central, receptors (J. J. Vanderhaegen and J. M. Crawley, Ann. N.Y. Acad. Sci., 1985, 448: 1-697).
Although interesting effects might be expected from the pharmacological stimulation of CCK receptors, no agent which stimulates these receptors is currently therapeutically available, mainly for reasons of poor bioavailability of CCK and derivatives thereof. In addition, it has even been shown that direct stimulation of CCK receptors was liable to lead to undesirable anxiogenic effects.
Other studies have been geared towards identifying the enzyme(s) responsible for the physiological inactivation of these endogenous neuropeptides and in particular CCK.
Accordingly, the involvement has been suggested of various enzymes which are thought to attack certain peptide bonds in the molecule CCK-8, such as, in particular, that of an acidic aminopeptidase, which is distinct from aminopeptidase A (Deschodt-Lanckman et al., Peptides, 1983, 4: 71), of enkephalinase and of an aminopeptidase (Matsas et al., FEBS Lett., 1984, 175: 124; Deschodt-Lanckman et al., Regul. Peptides, 1981, 2: 15), that of thiolpeptidases (Mc Dermott et al., Neurochem. Int., 1983, 5: 641; Durieux et al., Neuropeptides, 1986, 7: 1) as well as that of a metalloendopeptidase (Steardo et al., J. Neurochem., 1985, 45: 784).
More recently, Rose et al. (Proc. Natl. Acad., Sci. USA, 1988, 85: 8326; Neurosci., 1989, 29: 583) have suggested the involvement of a serine peptidase. They also observed that general reagents for the serine groups of proteins, such as diisopropyl fluorophosphorate (DFP) or elastase-inhibiting boronic acids or chloromethyl ketones (a family of serine peptidases) were capable of preventing the enzymatic degradation of endogenous CCK-8 released by depolarization of slices of brain.
However, these experiments did not allow the enzyme responsible to be identified and purified, and the compounds used in these experiments in vitro should not be considered as drugs, in particular on account of their toxicity, their absence of specificity and/or their poor bioavailability.
More recently still, an enzyme has been purified and identified from soluble extracts of human brain (Wilson C. et al., Neurochem. Res. 1993, 18(7), 743-749), this enzyme having much similarity with a protease known as tripeptidylpeptidase TPP II previously isolated from rat liver or from human erythrocytes (Balow R. M. et al., J. Biol. Chem., 1986, 261: 2409-2417; Tomkinson B. et al., Biochemistry 1991, vol. 30, 168-174). The 4611-bp sequence of a mouse TPP II has also been described (Tomkinson B., Biochem. J., 1994, vol. 304: 517-523), the coding sequence of which shows great homology with the abovementioned human TPP II.
This purified enzyme was shown to be capable of hydrolysing several neuropeptides including exogenous CCK-8, but neither its application in the inactivation of endogenous CCK-8 nor the possibility of obtaining CCK-type biological responses by inhibiting it had been either demonstrated or even suggested.
Thus, it is seen that there is a need in the prior art for specific, bioavailable, non-toxic chemical compounds which can be used as drugs for preventing the inactivation of these endogenous neuropeptides.
Moreover, a TPP II inhibitor of weak affinity has been described (Tomkinson et coll., Arch. Biochem. Biophys., 1994, 314: 276), but this inhibitor has an oligopeptidergic structure, which raises the idea that its bioavailability is low, especially orally, and that it therefore cannot constitute a drug, this use, incidentally, not having been considered by the authors.
The invention has now made it possible to achieve this objective by providing, in particular, a process for screening drugs which uses the isolated enzyme responsible, thus making it possible, depending on whether the said enzyme is or is not inhibited, to distinguish molecules liable to constitute effective drugs in the treatment of disorders or complaints involving endogenous neuropeptides, and in particular cholecystokinin.
The present invention also provides chemical compounds of formula defined below, which can be used to prevent the inactivation of endogenous neuropeptides, and thus satisfies the essential need existing in the prior art.
The inventors have prepared a pure membrane-derived tripeptidylpeptidase according to a process comprising the following steps:
i) preparation of membranes from brain (cerebral cortex), for example from rat brain;
ii) purification by high performance liquid chromatography/ies (HPLC);
iii) checking of the product obtained, by enzymatic reaction using a CCK substrate, for example the peptides CCK-8 (non-sulphated) of formula:
Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (SEQ ID NO: 7)
or CCK-5 of formula:
Gly-Trp-Met-Asp-Phe-NH2 (SEQ ID NO: 8)
Studying the specificity of this purified enzyme on a series of model substrates showed that it behaves like an aminotripeptidylpeptidase.
The inventors have carried out sequencing work. One fragment of the purified protein is highly similar to a protease known as tripeptydylpeptidase II isolated previously from human erythrocytes or from rat liver (Balxc3x6w R. M. et al., and Tomkinson et al.).
Using traditional molecular cloning methods, the inventors identified two distinct clones in a library of rat brain complementary DNA using two probes A and B of 350 and 380 bases respectively. They were obtained by a polymerase chain reaction (PCR) performed using the following primers:
SEQ ID No. 3 (probe A, sense primer): GACTGAGGAGCCCTTCCCTTTTCA
SEQ ID No. 4 (probe A, antisense primer): GCCTTAGGATAGAAGTCATAGCCA
SEQ ID No. 5 (probe B, sense primer): CCCTTTGTAGGAAAGGTTGTGCC
SEQ ID No. 6 (probe B, antisense primer): GAATACGCAATAATCGGGAGGATAC
Their sequencing indicated that the first clone is the rodent homologue of human tripeptidylpeptidase II (TPP II). On the other hand, in the second, the sequence differs in the 5xe2x80x2 part (starting from nucleotide 293).
The nucleotide sequence coding for the isolated protein is given by the identifiers (SEQ ID No. 1 and SEQ ID No. 2).
The sequence includes a hydrophobic segment of about twenty amino acids, indicating the existence of a transmembrane segment. Thus, although this protein is probably derived from the same gene as the above one by a process of alternative splicing, it presents itself as a serine ectopeptidase.
Thus, the subject of the present invention is a process for screening drugs by measuring the activity of the membrane-bound tripeptidylpeptidase II enzyme or homologue, by using a model substrate for this enzyme.
Tripeptidylpeptidase can be prepared in accordance with the abovementioned process.
According to another embodiment, brain membranes, prepared by simple centrifugation of a homogenate, are incubated in the presence of an aminotripeptidylpeptidase substrate (such as AAF-Amc) and potential inhibitors of the enzymatic activity thus revealed.
The term homologous enzyme is understood to refer both to tripeptidylpeptidases which might be genetically modified and membrane-free, especially soluble, tripeptidylpeptidases, such as the one mentioned above, isolated by Barlxc3x6w R. M. et al.
The inventors have been able to develop degradation inhibitors for endogenous neuropeptides, in particular CCK, by measuring the activity of the said enzyme in purified form, but also, more simply, in native form, on membrane preparations from tissues using a model substrate of TPP II.
Thus, the subject of the present invention is also chemical compounds corresponding to the general formula (I) below: 
in which:
R1 represents a hydrogen or a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen or a C1-C2 alkyl group;
at least one from among R1 and R2 representing a hydrogen;
n=0 or 1 and m=0 or 1 with n being different from m;
R and Rxe2x80x2 each independently represent a hydrogen or a C1-C2 alkyl group;
R3 represents a divalent radical consisting of a xe2x80x94(CH2)2xe2x80x94, xe2x80x94CH2xe2x80x94CH(cis.F)xe2x80x94 or xe2x80x94CH2xe2x80x94CH(CH2Ph)xe2x80x94 alkyl chain, of a unit 
where R6 represents H, F, OCH3 or OCH2Ph, 
where R8, R9 and R10 each represent a hydrogen or halogen atom, an O(C1-C4 alkyl), OCH2Ph, OH or C1-C4 alkyl group, including R6, (m) and (n) indicating the bond orientation with respect to the (CH2)n group (or to N if n=0) and to the (CRRxe2x80x2)m group (or to CHR4 if m=0)
R4 represents an amide group COxe2x80x94NHxe2x80x94R5 where R5 represents a hydrogen or a linear or branched C1-C6 alkyl, xe2x80x94(CH2)3xe2x80x94SCH3, xe2x80x94CH2Ph, xe2x80x94CH2C6H11, (CH2)3OH, 
xe2x80x83group.
Throughout the description, xe2x80x9cPhxe2x80x9d denotes an optionally substituted phenyl radical.
The compounds of formula (I) have several asymmetric centres and the invention thus covers the racemic mixtures as well as the various stereoisomers and mixtures thereof in any proportion.
The subject of the present invention is also chemical compounds corresponding to the general formula (Ixe2x80x2) below: 
in which:
R1represents a hydrogen or a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen or a C1-C2 alkyl group;
at least one from among R1 and R2 representing a hydrogen;
n=0 or 1 and m=0 or 1 with n being different from m;
R3 represents a divalent radical consisting of a xe2x80x94(CH2)2xe2x80x94, xe2x80x94CH2xe2x80x94CH(cis.F)xe2x80x94 or xe2x80x94CH2xe2x80x94CH(CH2Ph)xe2x80x94 alkyl chain, of a unit 
where R6 represents H, F, OCH3 or 
R4 represents an amide group COxe2x80x94NHxe2x80x94R5 where R5 represents a hydrogen or a linear or branched C1-C6 alkyl, xe2x80x94(CH2)3xe2x80x94SCH3, xe2x80x94CH2Ph, xe2x80x94CH2C6H11, 
xe2x80x83group.
According to a first preferred variant, the subject of the invention is compounds of formula (I) as defined above in which n and m are either equal to 0 or 1 with the proviso, however, that n is different from m.
According to a first aspect, the invention relates more particularly to a first group of these compounds in which R3 represents the divalent radical xe2x80x94(CH2)2xe2x80x94.
A sub-family according to this aspect includes compounds where R and Rxe2x80x2 each represent a hydrogen atom.
Compounds of this type are, especially, the compounds given in the examples below, Nos. 1 to 7 (prepared according to route 1); No. 8 (route 2); No. 9 (route 5); No. 10 (route 6).
Among the latter group of compounds, those in which R1 represents CH2CH3, (CH2)2CH3, (CH2)3CH3, CH (CH3)2, CH2CH(CH3)2 and CH(CH3)CH2CH3, R2, R and Rxe2x80x2 represent a hydrogen, R4 represents the amide group COxe2x80x94NHxe2x80x94R5 and R5 represents a hydrogen, are known.
The compound of formula (I) in which R1 represents CH2CH(CH3)2, R2, R and Rxe2x80x2 represent hydrogen, R3 is the divalent radical xe2x80x94(CH2)2xe2x80x94 and R5 represents CH2CH3 is also known.
The compound of formula (I) in which R1 represents CH3, R2, R and Rxe2x80x2 represent hydrogen, R3 is the xe2x80x94(CH2)2xe2x80x94 radical and R5 represents the unit 
is also known.
According to another aspect, the invention relates more particularly to a second group of these compounds in which R3 represents the cis-fluoroethylene unit xe2x80x94CH2xe2x80x94CH(cis.F)xe2x80x94 (substituted proline skeleton). One sub-family includes compounds in which R and Rxe2x80x2 are each a hydrogen, such as, for example, the compound of Example 11 (route 3) described below.
According to another aspect, the invention relates to a third group of these compounds in which R3 represents the benzylethylene unit xe2x80x94CH2xe2x80x94CH(CH2Ph)xe2x80x94 (substituted proline skeleton). One sub-family includes compounds in which R and Rxe2x80x2 are each a hydrogen, such as, for example, the compound of Example 12 (route 4) described below.
According to a second preferred variant, the subject of the invention is compounds of formula (I) as defined above in which n=0 and m=1.
According to one aspect of this variant, the invention relates to compounds in which R3 represents the unit 
where (n) and (m) indicate the bond orientation as above.
One sub-family includes compounds in which R and Rxe2x80x2 are each hydrogen, especially including the compound of Example 13 (route 3) described below.
According to a second particularly preferred aspect, the subject of the invention is compounds of formula (I) in which R and Rxe2x80x2 represent hydrogen and R3 represents the unit 
examples of which are, in particular, the compounds of Examples 14 to 17 (route 3); Examples 18 to 21 (route 7); described below.
According to another particularly preferred aspect of this second variant, the invention relates to compounds of formula (I) in which R3 represents the unit 
examples of which are, in particular, the compounds of Examples 24 to 26 (route 8) and of Example 27 (route 9); 28 (route 11); 29 and 30 (route 10); 31 and 32 (route 9); 33 and 34 (route 12); 35 (route 8); 38 (route 13) and 37 (route 14) described below.
This class of compounds includes compounds of formula (I) in which R3 represents the unit 
when R and Rxe2x80x2 each represent a hydrogen, two of the substituents R8, R9 and R10 then being a hydrogen.
According to another aspect of this second variant, the invention relates to compounds in which R3 represents the unit 
One sub-family includes compounds in which R and Rxe2x80x2 each represent hydrogen, such as, in particular, the compound of Example 22 (route 3) given below.
Lastly, according to a third variant, the subject of the invention is compounds of formula (I) in which n=and m=0 and R3 represents the unit 
such as the compound of Example 23 (route 4) described below.
The compounds of formula (I) in which R2 represents a hydrogen are also particularly preferred.
The inhibitory effect of these compounds was evaluated by measuring the membrane-bound TPP II activity and expressed in terms of their apparent dissociation constant Ki as described below.
The preferred compounds according to the invention are the following compounds:
2(S)-aminobutyryl-L-prolinamide;
L-valyl-L-proline n-hexylamide;
1-(2(S)-aminobutyryl)-L-proline 3-(methylthio)propylamide;
1-(2(S)-aminobutyryl)-L-proline n-pentylamide;
1-(2(S)-aminobutyryl)-L-proline n-butylamide;
1-(2(S)-aminobutyryl)-L-proline [2(S)-methyl]butylamide;
1-(2(S)-aminobutyryl)-L-proline n-propylamide;
1-(2(S)-aminobutyryl)-L-proline isobutylamide;
L-valyl-L-proline n-butylamide;
L-alanyl-L-prolyl-L-borovaline pinanediol ester;
L-alanyl-L-prolyldifluoro-L-borovaline borohydride;
1-(2(S)-aminobutyryl)-(4(S)-fluoro)-L-proline n-butylamide;
1-(2(S)-aminobutyryl)-(4(S)-benzyl)-L-proline n-butylamide;
2-(2(S)-aminobutyryl)-1,2,3,4-tetrahydro-3(S)-isoquinolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(S)-indolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(S)-indolinecarboxylic acid n-propylamide;
1-(2(S)-aminobutyryl)-2(S)-indolinecarboxylic acid methylamide;
1-(2(S)-aminobutyryl)-2(S)-indolinecarboxylic acid ethylamide;
1-(2(S)-aminobutyryl)-2(R/S)-(5-methoxy)indolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(R/S)-(6-methoxy)indolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(R/S)-(5-fluoro)indolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(R/S)-(5-benzyloxy)indolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(S)-[(3aS, 7aS)-perhydro]indolinecarboxylic acid n-butylamide;
2-(2(S)-aminobutyryl)-1(R/S)-isoindolinecarboxylic acid n-butylamide;
as well as the corresponding salts or hydrates thereof.
These compounds constitute preferred inhibitors according to the invention and have a constant Ki with respect to the tripeptidylpeptidase enzyme. according to the invention, of less than or equal to 1 xcexcM.
Other compounds that are most particularly preferred are compounds of formula (I) having an indoline skeleton, namely the following compounds:
1-(2(S)-aminobutyryl)-2(S)-indolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(S)-indolinecarboxylic acid n-propylamide;
1-(2(S)-aminobutyryl)-2(S)-indolinecarboxylic acid ethylamide;
1-(2(S)-aminobutyryl)-2(R/S)-(5-methoxy)indolinecarboxylic acid n-butylamide;
1-(2(S)-aminobutyryl)-2(R/S)-(5-fluoro)indolinecarboxylic acid n-butylamide;
as well as the corresponding salts or hydrates thereof.
These compounds are particularly preferred inhibitors having a constant Ki which does not exceed 0.02 xcexcM.
Other compounds that are most particularly preferred are also the compounds of formula (I) below having an indoline skeleton:
1-(L-valyl) -5-methoxyindoline-2(R/S)-carboxylic acid butylamide;
1-(L-alanyl)-5-methoxyindoline-2(R/S)-carboxylic acid butylamide;
1-(L-alanyl)-5-methoxyindoline-2(S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-4-methoxyindoline-2(R/S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-3,3-dimethylindoline-2(R/S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-3(R)-methylindoline-2(R)-carboxylic acid butylamide and 1-(2(S)-aminobutyryl)-2(S)-methylindoline-2(S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-3(R)-methylindoline-2(S)-carboxylic acid butylamide and 1-(2(S)-aminobutyryl)-3(S)-methylindoline-2(R)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-4-ethoxyindoline-2(S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-4,5-dimethoxyindoline-2(R/S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-5-hydroxyindoline-2(S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-5-hydroxyindoline-2(R/S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-5-methylindoline-2(R/S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)-5-chloroindoline-2(S)-carboxylic acid butylamide;
1-(2(S)-aminobutyryl)indoline-2(S)-carboxylic acid (3-hydroxy)propylamide;
as well as the corresponding salts or hydrates thereof.
The subject of the present invention is also processes for the preparation of the compounds of general formula (I) described above.
Thus, the invention relates more particularly to a process (route 1) for the preparation of a compound of general formula (I) defined above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen;
R and Rxe2x80x2 represent a hydrogen;
n=0 or 1 and m=0 or 1 with n being different from m and R3 represents xe2x80x94(CH2)2xe2x80x94; and
R4 represents COxe2x80x94NHxe2x80x94R5 
R5 represents a hydrogen or a linear or branched C1-C6 alkyl, xe2x80x94(CH2)3xe2x80x94Sxe2x80x94CH3 or xe2x80x94CH2Ph group;
characterized in that it comprises:
i) the formation of a compound of formula (III) 
xe2x80x83in which R1 and R2 have the meanings given above and X represents a protecting group, starting with a compound of formula (II) 
xe2x80x83which is esterified on its acid function with a group Y and in which X, R1 and R2 have the meanings given above, by reaction with L-proline;
(ii) amidation of the acid function of compound (III) with the appropriate amine R5NH2 
where R5 has the meaning given above, in order to form the derivative (IV) 
xe2x80x83which is protected on its primary amine function with the group X;
iii) removal of the group X from the derivative (IV), in order to obtain the desired compound (I).
The group Y preferably represents the unit 
Step i) is carried out in the presence of triethylamine and of water.
Moreover, in step ii), the mixed anhydride of the acid (III) and of isobutyl chloroformate is formed and is then reacted in situ with the amine R5NH2.
In the compounds prepared according to this route 1, R1 preferably represents an ethyl or isopropyl group.
The invention also relates to a process (route 2) for the preparation of a compound of general formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen or a methyl group;
R and Rxe2x80x2 represent a hydrogen;
n=0 or 1 and m=0 or 1 with n being different from m, and R3 represents a xe2x80x94(CH2)2xe2x80x94 group; and
R4 represents a group COxe2x80x94NHxe2x80x94R5 in which R5 represents a hydrogen or a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) formation of a compound of formula (IV) 
xe2x80x83in which R1, R2 and R5 have the meanings given above and X represents a protecting group, starting with a compound of formula (II) 
xe2x80x83which is esterified on its acid function with a group Y and in which R1, R2 and X have the meanings given above, by reaction with a prolineamide of formula (V) 
xe2x80x83in which R5 has the meaning given above;
ii) removal of the protecting group X from compound (IV) in order to obtain the desired compound (I).
The prolineamide (V) can be prepared by reacting L-proline 
which is protected on its amine function with a protecting group X and esterified on its acid function with a group Y, with the appropriate amine R5NH2.
The group Y preferably represents the unit 
In order to form compound (IV), the prolineamide thus obtained is reacted with compound (II), in the presence of water and triethylamine in a solvent such as tetrahydrofuran or dioxane.
In the compounds prepared according to this route 2, R1 preferably represents an ethyl or isopropyl group and R5 preferably represents a hydrogen or an n-butyl group.
The invention also relates to a process (route 3) for the preparation of a compound of general formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen;
R and Rxe2x80x2 represent a hydrogen;
n=0 and m=1 and R3 represents a group 
R4 represents a group COxe2x80x94NHxe2x80x94R5 in which R5 represents a hydrogen or a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) preparation of a compound of formula (X) 
xe2x80x83in which R1, R2, R3 and R4 have the meanings given above and X represents a protecting group, by reaction of a compound of formula (VIII) 
xe2x80x83with a compound of formula (IX) 
xe2x80x83in which R1, R2, R4 and X have the meanings given above;
ii) removal of the group X from the compound (X), in order to form the desired compound (I).
According to this route 3, compound (VIII) is prepared by amidation of the acid function of a compound (VI) 
which is protected on its amine function with a protecting group X and in which R3 has the meaning given above, with the appropriate amine R5NH2, in order to form the derivative (VII) 
in which R3 R4 and X have the meanings given above, followed by removal of the protecting group X.
For this, the mixed anhydride of the acid (VI) and of isobutyl chloroformate is advantageously formed in the presence of N-ethylmorpholine in tetrahydrofuran, and is then reacted in situ with the appropriate amine R5NH2. These conditions can also constitute those of step i) except that compound (VII) is reacted instead of the amine R5NH2.
In the compounds prepared according to this route 3, R1 preferably represents an ethyl group and R5 preferably represents a hydrogen or a methyl, ethyl, n-propyl or n-butyl group.
The invention also relates to a process (route 4) for the preparation of a compound of general formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen;
n=1 and m=0 and R3 represents a xe2x80x94CH(CH2Ph)xe2x80x94CH2xe2x80x94 group or 
R4 represents an amide group COxe2x80x94NHxe2x80x94R5 in which R5 represents a hydrogen or a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) production of a compound (XII) 
xe2x80x83which is protected on its primary amine function with a protecting group X and in which R1, R2 and R3 have the meanings given above, by reaction of the compound of formula (IX) 
xe2x80x83with the compound of formula (XI) 
xe2x80x83in which R1, R2 and R3 have the meanings given above and X represents a protecting group;
ii) hydrolysis of the ester function of compound (XII) thus obtained, in order to form the compound of formula (XIII) 
xe2x80x83in which R1, R2, R3 and X have the meanings given above;
iii) amidation of the acid function of compound (XIII) using the appropriate amine R5NH2, in order to form the derivative of formula (XIV) 
xe2x80x83R1, R2, R3, R5 and X have the meanings given above;
iv) removal of the protecting group X from the compound (XIV) in order to form the desired compound (I).
Step ii) of hydrolysis is advantagelously carried out in the presence of sodium hydroxide in methanol.
In step iii), the mixed anhydride of the acid (XIII) and of isobutyl chloroformate is formed and is then reacted in situ with the appropriate amine R5NH2.
In the compounds prepared according to this route 4, R1 preferably represents an ethyl group and R5 preferably represents a hydrogen or an n-butyl group.
The invention also relates to a process (route 5) for the preparation of a compound of general formula (I) given above, in which:
R1 represents a methyl group;
R2 represents a hydrogen;
R and Rxe2x80x2 represent a hydrogen;
n=0 or 1 and m=0 or 1 with n being different from m, and R3 represents the xe2x80x94(CH2)2xe2x80x94 group; and
R4 represents an amide group COxe2x80x94NHxe2x80x94R5 in which R5 represents the unit 
xe2x80x83characterized in that it comprises:
i) amidation of the acid function of the compound of formula (XIX) 
xe2x80x83which is protected on its primary amine function with a protecting group X and in which R1 and R2 have the meanings given above, using the amine (XVIII) 
xe2x80x83in order to form the derivative (XX) 
in which R1, R2, R5 and X have the meanings given above;
ii) removal of the protecting group X from compound (XX) in order to obtain the desired compound (I).
Step i) is advantageously performed using isobutyl chloroformate, in order to form the mixed anhydride of the acid XIX, in the presence of N-ethylmorpholine in tetrahydrofuran.
Step ii) can, itself, be carried out using trifluoroacetic acid in methylene chloride.
The amine (XVIII) is prepared
i) by reaction of isopropylboronic acid with (+)-pinanediol in ether, in order to obtain the derivative (XV) 
ii) by reaction of compound (XV) with butyllithium and methylene chloride, followed by zinc chloride ZnCl2 in tetrahydrofuran, at low temperature, in order to for m derivative (XVI) 
iii) substitution of the chlorine in compound (XVI) using butyllithium and hexamethyldisilazane in tetrahydrofuran at low temperature, in order to form compound (XVII) 
iv) liberation of the amine function in compound (XVII) in order to obtain the desired compound (XVIII).
Isopropylboronic acid is obtained by addition of triethyl borate to isopropylmagnesium chloride in ether at low temperature.
Step iv) mentioned above is advantageously performed using trifluoroacetic acid in pentane.
The invention also relates to a process (route 6) for the preparation of a compound of general formula (I) given above, in which:
R1 represents a methyl group;
R2 represents a hydrogen;
R and Rxe2x80x2 represent a hydrogen;.
n=0 or 1 and m=0 or 1 with n being different from m, and R3 represents a xe2x80x94(CH2)2xe2x80x94 group; and
R4 represents an amide group COxe2x80x94NHxe2x80x94R5 in which R5 represents (CH3)2xe2x80x94CHxe2x80x94BF2;
characterized in that it comprises:
i) removal of the pinane unit from the compound of formula (I) in which R1, R2, n, m, R3 and R4 have the meanings given above and R5 represents 
xe2x80x83by the action of boron trichloride in methylene chloride, followed by hydrolysis, in order to obtain derivative (XXI) 
ii) reaction of compound (XXI) with hydrofluoric acid in order to form the desired compound (I).
The invention relates to a process (route 7) for the preparation of a compound of general formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen;
R and Rxe2x80x2 represent a hydrogen;
n=0 and m=1 and R3 represents the unit 
in which R6 represents an OCH3, OCH2Ph or F group; and
R4 represents an amide group COxe2x80x94NHxe2x80x94R5 in which R5 represents a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) formation of the amide of formula (XXXXIV) 
xe2x80x83starting with the ester of formula (XXXXIII) 
in which formulae R6 has the meaning given above, by reaction with the appropriate amine R5NH2;
ii) reaction of compound (XXXXIV) with the compound of formula (IX) 
in which R1 and R2 have the abovementioned meanings and X represents a protecting group, in order to form the compound of formula (XXXXV) 
in which R1, R2, R5, R6 and X have the meanings given above;
iii) removal of the group X in order to form the desired compound (I).
Step ii) is advantageously carried out in the presence of bis(2-oxo-3-oxazolidinyl)phosphinyl chloride and triethylamine in methylene chloride.
As regards the methyl ester (XXXXIII), it can be obtained
i) by the action of sodium nitrite in the presence of hydrochloric acid, on the compound of formula (XXXIX) 
xe2x80x83in order to form the compound of formula (XXXX) 
in which formulae R6 has the meaning indicated above
ii) by addition of ethyl 2-methylacetoacetate to compound (XXXX) thus obtained, in the presence of sodium nitrite in ethanol, in order to form the compound of formula (XXXXI) 
in which R6 has the abovementioned meaning;
iii) by cyclization in acidic medium, in order to form the ethyl ester (XXXXII) 
iv) by exchange starting with the ethyl ester (XXXXII) thus obtained, in the presence of magnesium in ethanol.
The ethyl ester undergoes an ester exchange and a reduction in the presence of magnesium and methanol in order to lead to the methyl ester (XXXXIII).
In the compounds prepared according to this route 7, R1 preferably represents an ethyl group and R5 preferably represents an n-butyl group.
The invention relates to a process (route 8) for the preparation of a compound of general formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2, R and Rxe2x80x2 each represent a hydrogen;
n=0 and m=1, and R3 represents the unit 
in which R8 and R10 represent a hydrogen and R9 represents a group O(C1-C4 alkyl) or C1-C4 alkyl
R4 represents an amide group COxe2x80x94NHxe2x80x94R5 in which R5 represents a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) formation of the amide of formula (49) 
xe2x80x83starting with the ester of formula (48) 
in which R9 and R5 have the meaning given above, by reaction with the appropriate amine R5NH2;
ii) reaction of compound (49) with the compound of formula (IX) 
in which R1 and R2 have the abovementioned meanings and X represents a protecting group, in order to form the compound of formula (50) 
in which R1, R2, R9 and X have the meanings given above;
iii) removal of the group X in order to form the desired compound (I).
Step ii) is advantageously carried out in the presence of bis(2-oxo-3-oxazolidinyl)phosphonyl chloride and triethylamine in methylene chloride.
The methyl ester (48) can be obtained from the corresponding acid (46) 
in which R9 has the meaning given above
i) by treatment in ethanol or methanol with concentrated sulphuric acid, in order to lead to the corresponding ester (47) 
in which R9 is as defined above
ii) after which, compound (47) is treated with magnesium in methanol.
In the compounds prepared according to this route 8, R1 preferably represents a CH3, C2H5 or (CH3)2CH group, R9 preferably represents an OCH3 or CH3 group and R5 preferably represents an n-butyl group.
The subject of the invention is also a process (route 9) for the preparation of a compound of general formula (I) given above, in which
R1 represents a linear or branched C1-C4 alkyl group;
R2, R and Rxe2x80x2 each represent a hydrogen;
n=0 and m=1, and R3 represents the unit 
in which R8 and R9 represent a hydrogen or an O(C1-C4 alkyl) group, it not being possible for R8 and R9 simultaneously to represent a hydrogen, and R10 represents a hydrogen
R4 represents an amide group CONHR5 in which R5 is a linear or branched C1-C6 alkyl group,
characterized in that it comprises:
i) formation of compound (54) 
xe2x80x83by reaction of the corresponding aldehyde (53) 
in which R8, R9 and R10 are as defined above with ethyl azidoacetate (52) 
ii) cyclization of compound (54) in order to lead to compound (55) 
in which R8, R9 and R10have the meanings given above
iii) formation of methyl ester (56) 
in which R8, R9 and R10have the meanings given above
starting with compound (55) in the presence of magnesium in methanol
iv) reaction of the ester (56) obtained with the appropriate amine R5NH2 in order to form the amide (57) 
in which R8, R9 and R10 are as defined above
v) reaction of compound (57) with the compound of formula (IX) 
in which R1 and R2 have the abovementioned meaning and X represents a protecting group, in order to form compound (58) 
in which R1, R2, R5, R8, R9, R10 and X have the meaning given above,
vi) removal of the group X in order to form the desired compound (I).
Compound (52) can be obtained by the action of sodium azide on ethyl bromoacetate in acetonitrile.
According to step i), sodium ethoxide is first formed and compounds (52) and (53) are then reacted.
The cyclization step ii) is advantageously carried out by refluxing in toluene.
Step v) is advantageously carried out in the presence of bis(2-oxo-3-oxazolidinyl)phosphonyl chloride and triethylamine in methylene chloride.
In the compounds prepared according to this route 9, R1 preferably represents the CH2CH3 group, R8 an OCH3 or OC2H5 group and R9 a hydrogen, or alternatively R8 and R9 both represent an OCH3 group, and R5 preferably represents an n-butyl group.
The subject of the invention is also a process (route 10) for the preparation of a compound of formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen;
one of the substituents R or Rxe2x80x2 represents a hydrogen and the other a C1-C2 alkyl group;
n=0 and m=1 and R3 represents the unit 
with R8, R9 and R10 representing a hydrogen
R4 represents an amide group CONHR5 in which R5 is a linear or branched C1-C6 alkyl group,
characterized in that it comprises:
i) formation of compound (59) 
in which R and Rxe2x80x2 are as defined above by reaction between phenylhydrazine and 2-ketobutyric acid in acidic medium
ii) formation of compound (60) from the compound (59) obtained 
in which R and Rxe2x80x2 are as defined above in the presence of magnesium in methanol,
iii) formation of the amide (61) corresponding to compound (60) by reaction with the appropriate amine R5NH2 
in which R and Rxe2x80x2 are as defined above
iv) separation of the cis isomers (61a), on the one hand, and the trans isomers (61b), on the other hand, of compound (61) 
v) reaction, respectively, of compounds (61a) and (61b) with compound (IX) 
in which R1 and R2 have the meaning given above and X represents a protecting group, in order to form mixtures (62a) and (62b) respectively 
in which R1, R2, R, Rxe2x80x2 and R5 have the meaning given above
vi) removal of the protecting group X, leading to the desired compound (I) in the form of cis pairs (63a) and trans pairs (63b), respectively.
Step v) is advantageously carried out in the presence of bis(2-oxo-3-oxazolidinyl)phosphinyl chloride and triethylamine in methylene chloride.
In the compounds prepared according to this route 10, R1 preferably represents the CH2CH3 group, R or Rxe2x80x2 represents the CH3 group (the other substituent being a hydrogen) and R5 preferably represents the n-butyl group.
The subject of the invention is also a process (route 11) for the preparation of a compound of formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2 represents a hydrogen;
R and Rxe2x80x2 each represent a C1-C2 alkyl group, which may be identical or different;
n=0 and m=1 and R3 represents the unit 
in which R8, R9 and R10 each represent a hydrogen;
R4 represents an amide group CONHR5 in which R5 is a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) formation of compound (65) of the following formula: 
by reaction of phenylhydrazine with ethyl 2-oxo-3-methylbutanoate (64)
ii) cyclization of compound (65) in acidic medium, in order to form compound (66) below: 
iii) hydrogenation of compound (66), leading to compound (67) below 
iv) formation of the corresponding amide (68) by the action of LiNHR5 in which R5 has the abovementioned meaning 
v) reaction of compound (68) with the compound of formula (IX) 
in which R1 and R2 have the meaning given above and X is a protecting group, in order to form compound (69) 
vi) removal of the group X in order to form the desired compound (I).
Route 11 is illustrated above in the case where R and Rxe2x80x2 represent a methyl group, but R and Rxe2x80x2 can each represent an alkyl group, which may be identical or different. In this case, step i) is carried out with the appropriate compound, in particular a compound (64) 
Step i) is advantageously carried out at a temperature of about 60xc2x0 C. in toluene.
Step v) is advantageously carried out in the presence of bis(2-oxo-3-oxazolidinyl)phosphinyl chloride and triethylamine in methylene chloride.
Ethyl 2-oxo-3-methylbutanoate (64) can be obtained from diethyl oxalate with isopropylmagnesium chloride in ether at low temperature.
In the compounds prepared according to this route 11, R1 preferably represents an ethyl group and R5 preferably represents an n-butyl group.
The invention also relates to a process (route 12) for the preparation of a compound of formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2, R and Rxe2x80x2 each represent a hydrogen;
n=0 and m=1, and R3 represents the unit 
in which R9 represents an OH group and R8 and R10 both represent a hydrogen;
R4 represents an amide group CONHR5 in which R5 is a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) formation of compound (71) 
in which R9 represents an OCH2Ph group, R5 has the meaning given above and X represents a protecting group, by reaction of compound (70) 
in which R5 is as defined above, with a compound (IX) 
in which R1 and R2 have the meaning given above and X represents a protecting group,
ii) removal of the groups CH2Ph and X from compound (71) in order to form the desired compound (I).
Step i) is advantageously carried out in the presence of dicyclohexylcarbodiimide in methylene chloride.
Compound (70) can be obtained by the process described above (route 7).
In the compounds prepared according to this route 12, R1 preferably represents an ethyl group and R5 an n-butyl group.
The invention also relates to a process (route 13) for the preparation of a compound of formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2, R and Rxe2x80x2 each represent a hydrogen;
n=0 and m=1, and R3 represents the unit 
in which R9 represents a halogen atom and R8 and R10 each represent a hydrogen
R4 represents an amide group CONHR5 in which R5 is a linear or branched C1-C6 alkyl group;
characterized in that it comprises:
i) formation of the methyl ester (73) 
xe2x80x83from the corresponding acid (72) 
in which R9 is as defined above, with concentrated sulphuric acid in methanol,
ii) formation of compound (74) 
in which R9 is as defined above, starting with compound (73), with magnesium in methanol,
iii) formation of the corresponding amide (75) by reaction with the appropriate amine R5NH2 
in which R5 and R9 are as defined above
iv) reaction of compound (75) with a compound (IX) 
in which R1 and R2 have the meanings given above and X represents a protecting group, in order to form compound (76) 
in which R1, R2, R5, R9 and X have the meanings given above,
v) removal of the group X in order to form the desired compound (I).
Step iv) is advantageously carried out in the presence of bis(2-oxo-3-oxazolidinyl)phosphinyl chloride and ethylamine in methylene chloride.
In the compounds prepared according to this route 13, R1 preferably represents an ethyl group and R5 preferably represents an n-butyl group.
Lastly, the subject of the invention is a process (route 14) for the preparation of a compound of formula (I) given above, in which:
R1 represents a linear or branched C1-C4 alkyl group;
R2, R and Rxe2x80x2 each represent a hydrogen;
n=0 and m=1, and R3 represents the unit 
in which R8, R9 and R10 each represent a hydrogen;
R4 represents an amide group CONHR5 in which R5 is a (CH2)3OH group,
characterized in that it comprises:
i) reaction of the methyl ester of indoline-2S-carboxylic acid with a compound of formula (IX) 
in which R1 and R2 have the meaning given above and X represents a protecting group, in order to form compound (77) below 
in which R1, R2 and X are as defined above
ii) formation of amide (78) 
in which R1 and R2 have the above meaning starting with compound (77), by the action of 3-hydroxypropylamine in methanol
iii) removal of the group X in order to form the desired compound (I).
The methyl ester of indoline-2S-carboxylic acid can be prepared from indoline-2S-carboxylic acid with methanol and concentrated sulphuric acid.
Step ii) is advantageously carried out in the presence of bis(2-oxo-3-oxazolidinyl)phosphinyl chloride and triethylamine in methylene chloride.
In the compounds prepared according to this route 14, R1 preferably represents an ethyl group.
In all of the processes described above, formation of the acid anhydrides can be carried out by the standard methods, advantageously using isobutyl chloroformate in the presence of N-ethylmorpholine in tetrahydrofuran.
Moreover, according to the invention, X or Xxe2x80x2 represents a standard protecting group which is capable of protecting the desired function in a given molecule without affecting the other functions in that molecule, such as, in particular, the benzyl, benzyloxycarbonyl or tert-butoxycarbonyl groups. These groups are introduced by standard methods that are well known to those skilled in the art.
This is likewise the case for the deprotection steps, which are also carried out by methods known per se such as, in particular, acid hydrolysis or catalytic hydrogenation, in particular in the presence of Pd/C.
As regards the group Y, it is a radical capable of esterifying an acid function without modifying the other functions in the molecule, and constituting a good leaving group in order to be readily eliminated in a subsequent step. It is preferably the succinimide ester.
The Applicant""s research has shown that the compounds of formula (I) have many therapeutic properties and in particular in the field of treating disorders or complaints which involve inactivation or excessive degradation (or which may be treated by delaying the physiological degradation) of endogenous neuropeptides, such as, in particular, disorders or complaints associated with inactivation of CCK.
Thus, the subject of the present invention is also the use of a compound of formula (I) as a drug for treating, in man or animals, in particular, eating, mood, cognitive or motor disorders, in particular anorexia, schizophrenia, Parkinson""s disease and depression, as well as disorders of gastrointestinal transit such as irritable bowel syndrome, bulimia or pathological obesity conditions.
The subject of the present invention is also the use of a compound of formula (I) to prepare a drug intended for treating, in man or animals, complaints or disorders brought about by the physiological degradation of endogenous neuropeptides, in particular that of CCK.
The subject of the present invention is, in particular, the use of compounds of formula (I) to prepare a drug intended for treating disorders or complaints as mentioned above.
Compounds of the formula (I) which are useful as drugs may be administered in a physiologically acceptable vehicle.
Thus, the subject of the present invention is also pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula (I) in combination with a physiologically acceptable vehicle.
The subject of the invention is also a process for treating, in man or animals, complaints or disorders associated with excessive degradation (or which may be treated by delaying the physiological degradation) of endogenous neuropeptides, in particular disorders or complaints associated with the inactivation of CCK, in particular the disorders given as examples above, comprising the administration of a pharmaceutical composition as defined above.
The subject of the present invention is also the use of the compounds of formula (I) as inactivation inhibitors for endogenous neuropeptides and in particular CCK.
The inventors have observed that, in general, among the compounds of formula (I), those having an L,L chirality (that is to say S,S according to the Ingold-Kahn-Prelog nomenclature) are the most active. In the examples given below, the configuration of the other optically active carbons is also indicated for the compounds of formula (I) having more than two asymmetric centres.
In all the abovementioned uses, it is preferred to choose compounds according to the invention that have a Ki constant less than or equal to 1 xcexcM, such as those given as examples and, in an entirely preferable manner, the compounds of formula (I) having an indoline skeleton.
According to the invention, all the abovementioned uses also comprise that of the compounds of formula (I) which are already known, as indicated above, that is to say the compounds of formula (I) in which R2, R and Rxe2x80x2 represent a hydrogen, n=0 and m=1 or n=1 and m=0, R3 represents the xe2x80x94(CH2)2xe2x80x94 divalent radical, R4 represents an amide group CONHR5, R1 represents a CH2CH3, (CH2)2CH3, (CH2)3CH3, CH(CH3)2, CH2CH(CH3)2 or CH(CH3)CH2CH3 group with R5 representing a hydrogen, R1 represents CH2CH(CH3)2 with R5 representing a CH2CH3 group and R1 represents CH3 with R5 representing the unit 
Other advantages and characteristics of the present invention will become apparent on reading the experimental results which follow, in particular of a method of isolation and characterization of the tripeptidylpeptidase according to the invention, as well as the preparation examples given by way of non-limiting illustration.
The inventors used rat brain (cerebral cortex) membranes as starting material and non-sulphated CCK-8 (Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2) and CCK-5 (Gly-Trp-Met-Asp-Phe-NH2) peptides as substrates, the characteristic products of the reaction (CCK-5 and Gly-Trp-Met) being measured fluorimetrically after isolation by high performance liquid chromatography (HPLC) according to Camus et al., (Neurosci., 1989, 29: 595).
The subsequent steps of HPLC purification used are described in Table I below, which also indicates the purification factors obtained using CCK-8 as substrate.
From the second stage of purification onwards, the chromatography profile contains only one peak of enzymatic activity, on which is superimposed the peak obtained by measuring the hydrolysis of CCK-5, thereby indicating that only one enzyme is responsible for the two cleavages of the molecule CCK-8.
After the final step of purification, gel electrophoresis on sodium dodecyl sulphate (SDS) indicates a single band irreversibly labelled with 3H-DFP, at an apparent mass of 135 kDa.
Study of the specificity of the purified enzyme on a series of model substrates showed that it behaves like an aminotripeptidylpeptidase, capable, in particular, of hydrolysing Ala-Ala-Phe-p-nitroanilide or Ala-Ala-Phe-amidomethylcoumarin (A-AP-Amc) fragments, releasing nitroaniline or aminomethylcoumarin.
Preparation Examples for Compounds of Formula (I)